The production of recombinant proteins in eukaryotic cells may result in the co-purification of related host cell proteins. The production of human tissue plasminogen activator (t-PA) in Chinese hamster ovary (CHO) cells may therefore result in the co-purification of Chinese hamster plasminogen activator(s) (PA) produced by CHO cells (CHO-PA).
Human t-PA is an extremely important new biological pharmaceutical agent shown to have great promise in the treatment of vascular diseases due to its high specificity and potent ability to dissolve blood clots in vivo. Accordingly, t-PA has been hailed by medical science as one of the most impressive new agents of recent history for the treatment of vascular occlusive disease, and in particular, heart disease. For these and other reasons, t-PA will likely revolutionize the clinical management of serious vascular disease.
Human t-PA protein, as well as the underlying gene sequences which code for it, has been the subject of numerous scientific disclosures over the previous few years. For example, its isolation from natural sources has been described by Rijken et al., J. Biol Chem. 256, 7035 (1981) and European Patent Application Publication No. 041766. Moreover, a patent and various patent applications have been published detailing the structure and isolation of t-PA from recombinant sources (see, e.g., UK Patent 2,119,804; and European Patent Application Publication No. 093619).
Monoclonal antibodies which bind to human t-PA but do not bind to swine t-PA have been made, for example European Patent Application 190711 relates to monoclonal antibodies which are specific to human tissue plasminogen activator obtained from a tissue cultured liquor of normal human tissue derived cells. Use of these monoclonal antibodies in the purification of human t-PA and in an assay for human t-PA, are disclosed. Monoclonal antibodies X-21 and X-23 did not cross react with urokinase or plasminogen activator extracted from swine heart.
European Patent Application 210870 discloses an affinity reagent containing an immobilized Kunitz inhibitor which is produced in the seeds of Erythrina latissima and other Erythrina plants. The affinity reagent is said to be capable of separating human cell derived t-PA and host cell derived plasminogen activator from each other.
Verheijen et al., EMBO Journal, Vol. 5, No. 13, pp 3525-3530 (1986) disclose the presence of CHO-PA when expressing human t-PA in CHO cells.
It is an object of the subject invention to provide a method of purifying recombinant protein, e.g. human t-PA to a very high degree by substantial reduction of the corresponding endogenous protein of the host cell, e.g. Chinese hamster plasminogen activator.
It is a further object of the invention to provide monoclonal antibodies to Chinese hamster plasminogen activator.
It is a further object of the invention to provide an assay for Chinese hamster plasminogen activator.
Other objects, features and characteristics of the present invention will become more apparent upon consideration of the following description and the appended claims.